Abstract:Aminoacyl-tRNA synthetases are key enzymes in protein biosynthesis that catalyze aminoacylation of their cognate tRNA. During their long evolution, these ancient enzymes incorporated new domains by insertioos or fusions to the class-defining catalytic core to attend more functions. Recently, fragments of the closely related human tyrosyl- and tryptophanyl-tRNA synthetases were discovered to be active in angiogenesis signaling pathway. One synthetase fragment has proangiogenic activity, while the other is antiangiogenic. These two tRNA synthetases link protein synthesis to a major cell-signaling pathway in the given mammalian cells. The results with animals suggest that therapeutic applications for many human diseases such as neovascular eye disease and tumor are possible with these tRNA synthetases.

Abstract:Circadian photoreceptor in mammals is a kind of specific retinal ganglion cells which are light sensitive. Melanopsin has been proposed as an important photoreceptive molecule for the mammalian circadian system. Circadian photoreceptors are functionally characterized by the direct sensitivity to light with broad spectrum and the relatively high stability. The major circadian pacemaker in the hypothalamic suprachiasmatic nucleus is entrained to the light/dark cycles from the outside world by circadian photoreceptors.

Abstract:Three types of galanin receptor (GalR1, GalR2, GalR3) have been cloned to date, and identified to be G-protein coupled receptors. These three galanin receptors show differences in their amino acids sequences, pharmacological properties, as well as in the second messenger system. Activation of GalR1/3 induces inhibition on adenylyl cyclase and to activate K⁺ channels, while activation of GalR2 leads to the stimulation of phospholipase C and intracellular Ca²⁺ mobilization. The distribution of the three galanin receptors in human, rats and mice have been investigated by Northern blot, RT-PCR and in situ hybridization. The different distribution of the galanin receptor indicates that galanin participates a broad range of physiological activities.

Abstract:MicroRNAs (miRNA) belong to a novel large family of short single-stranded conserved noncoding regulatory RNAs that include the small temporal RNAs lin4 and let7. They exhibit a diversity in sequence, structure, abundance, and expression profile. There are similarities and differences between miRNAs and another class of RNAs called short interfering RNAs (siRNA). The recent advance of miRNAs is reviewed.

Abstract:Genetic vaccines were introduced less than a decade ago, but have already applied to a wide range of that fight off infectious, immunological, malignant diseases, and some genetic vaccines have been in clinical trials. Tumor genetic vaccines can break the immune tolerance, activate the immunogenicity, and induce the humoral and cellular responses to tumor cells. The anti-tumor genetic vaccines have proved to be effective prophylactic and curative vaccination against tumor. Progress in anti-tumor genetic vaccines is very rapid, including tumor-associated-antigens-based completed, epitope, idiotope determinants DNA vaccines, fusion DNA vaccines, RNA self-replicating vaccines, dendritic cell-based tumor vaccines etc. Meanwhile, the molecular mechanisms and the problems of the anti-tumor genetic vaccines also attract the scientists in this field.

Abstract:The direct information of the surface structure change of purple membrane under high pH was obtained. The UV-VIS spectra showed that BR was totally denatured and lost its retinal under the pH 12.6. It was found by atom force microscope that the crystalline lattice of purple membrane was broken down at this pH. The typical and non-typical “Island” structures emerged as the BR molecules assemble irregularly in purple membrane.

Abstract:In order to study the regulation of E-cadherin by protein kinase B (PKB), wild type SMMC 7721 hepato-carcinoma cells and a Gag-PKB/SMMC 7721 cell line where PKB activity is markedly increased compared with control cells were used. Interestingly, increasing PKB activity via insulin stimulation or Gag-PKB transfection does not enhance the E-cadherin in the level of mRNA and that of protein by using Northern blot and Western blot analysis, but markedly drives E-cadherin protein to cell surface by using flow cytometry analysis and immunofluorescence analysis localization of E-cadherin, which resulted in the increase of cell aggregation and the inhibition of cell apoptosis mostly via E-cadherin. Therefore, new evidences that elevation of PKB activity could drive functional E-cadherin molecule to cell surface are provided.

Abstract:BRD7 gene is a novel candidate tumor suppression gene associated with NPC. In order to study the function of BRD7, BRD7 was introduced into HNE1 cells by liposome transfection. After staining and image analysis, the ten differential expression spots which up-regulated in BRD7 transfected cells were isolated and identified by two-dimensional polyacrylamide gel eletrophoresis(2D PAGE) and MALDI-TOF. These proteins included argininosuccinate lyase, TSA, proteaseome activator 28 beta subunit, metalloproteinase inhibitor-2 precursor, which involved in cell cycling, transcription regulation,metabolism and so on. The results indicated that the BRD7 gene may play effect on NPC cells by up-regulating the expression of these proteins.

Abstract:In order to investigate the potential roles of Forkhead Box c2（Fox c2）in cardiovascular development, mice lacking Fox c2 locus were produced by targeted mutation and the developmental anomalies in the aortic arch were found. Mice homozygotes for the mutation(Fox c2^－/－) died embryonically from E12.5 (embryo days, E) to term. Although some of the homozygous mutants were born with abnormalities of the aortic arch, all of them died within 24 h after birth. Fox c2^－/－ homozygous mutants all displayed the Type B or Type C of interrupted aortic arch, which is the same as human congenital cardiovascular anomalies. Mice heterozygous for the mutation developed normally. In situ hybridization analysis on E10.5 embryos showed that Fox c2 mRNA expressed at the third, fourth and sixth arch arteries strongly. However, left fourth arch arteries disappeared at E12.5 gradually. These results suggest that the Fox c2 plays indispensable role in the remodeling of left fourth arch arteries during the formation of aortic arch.

Abstract:The interaction of human serum albumin with divalent nickel ion was studied by equilibrium dialysis, isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and circular dichroism (CD) in 30 mmol/L Tris buffer, pH=7.0. There is a set of 8 identical binding sites for nickel binding on the protein at two temperatures of 300 K and 310 K. The cooperativity in the binding is observed at 310 K. The Hill coefficients at 300 K and 310 K are 0.97 and 1.25, respectively. The interaction between nickel ions and HSA is exothermic. A value of －36.5 kJ for enthalpy of interaction (1∶1 stoichiometry) was obtained. The secondary structure of HSA dose not show any change during the binding nickel ions process. However, the tertiary structure of the protein changes, which shows the existence of two natives like states.

Abstract:The pIRES2-EGFP eukaryotic expression vectors carrying two kinds of reconstructed human caspase-8 genes with rearrangement subunits were transfected into HeLa cells. The expression and pro-apoptotic effects of reconstructed human caspase-8 gene on HeLa cells were analyzed by indirect fluorescent staining, immunohistochemical staining and electronic microscope etc.The results show that expression of the two human caspase-8 can induce HeLa cells apoptosis effectively.

Abstract:The contamination of chloroplast and mitochondrial DNAs was a serious problem during genome sequencing of rice (Oryza sativa L.ssp.indica) by whole-genome shotgun strategy. Pulse field gel electrophoresis (PFGE) was utilized to purify rice genomic DNA, which could efficiently remove the organelles (chloroplast and mitochondrion) DNA and reduce the contamination ratio from 3% to 0.2%. At the same time, the rice DNAs yielded from yellow seedlings and green seedlings were compared, and the differences between HB method and NIB method in high molecular weight(HMW) DNA isolation were also studied. Finally, a set of methods for obtaining the whole and highly pure rice genomic DNAs were proposed, which included culturing rice yellow seedling; isolating, embedding and lysising rice nuclei, purifying and recovering rice genomic DNAs in low melting point (LMP) agarose gel by PFGE. Ultrasonic treatment on HMW DNAs in the melting LMP gel at 38℃ was reported at first time, it facilitated to obtain the desired DNA fragments for construction of shotgun library and gradient libraries.

Abstract:It has been reported that activation of potassium channel is involved in the apoptosis of hippocampal neurons induced by in vivo ischemia and in vitro hypoxia. Recently, cell shrinkage is proposed as an early prerequisite to apoptotic events leading to cell death. To understand the mechanism underlying potassium channel-mediated neuronal apoptosis, the temporal changes in neuronal cell body volume and the involvement of potassium channel in the apoptotic volume decrease were examined in a model of staurosporine (STS)-induced apoptosis of cultured hippocampal neurons. Nonselective potassium channel blocker tetraethylammonium (TEA) or raising extracellular K⁺ concentration significantly prevented STS-induced neuronal cell death. A similar neuroprotection was also observed by treatment with the selective high-conductance calcium-activated potassium channel (BK) blockers iberiotoxin and paxilline. These results indicate that potassium channels, especially BK channels, contribute to STS-induced neuronal apoptosis. Moreover, STS induced an early cell body volume decrease and this cell shrinkage was completely blocked by TEA or high extracellular K⁺. It is suggested that potassium efflux may be involved in the apoptotic volume decrease, which is probably one of the mechanisms underlying mediation of neuronal apoptosis by potassium channel.

Abstract:It has been shown that intracellular Ca²⁺ in hippocampal CA1 neurons is elevated during ischemia and at early period following reperfusion. This Ca²⁺ overload has been suggested to be involved in ischemic brain damage. In normal CA1 neurons, the major mechanism allowing Ca²⁺ entry from the extracellular compartment is the opening of voltage-gated Ca²⁺ channels. The aim of the present study was to explore whether L-type calcium channel in hippocampal CA1 neurons changed at early period of reperfusion after ischemia. Transient forebrain ischemia in a duration of 15 min was induced by the use of the 4-vessel occlusion method in rats. Single L-type calcium currents were recorded in cell-attached patches of actually dissociated hippocampal CA1 neurons. After ischemia, average total patch current of L-type Ca²⁺ channels significantly increased in CA1 neurons when compared with that of control. This ischemia-induced enhancement in channel function was due to a higher channel open probability. Further analysis of single channel kinetics showed a prolonged open time and an increased opening frequency in postischemic channels. It is suggested that the functional enhancement in L-type calcium channels may partially account for the postischemic increase in intracellular Ca²⁺ concentration of CA1 neurons following ischemia.

Abstract:The effect of antisense RNA for urokinase receptor(uPAR) on inhibition of invasiveness of human lung giant cancer cell lines 95D was observed. 500bp fragment of uPAR cDNA between －46bp～+454bp was amplified, and recombined into plasmid pAdeno-X. The recombinant vector was named pAdeno-X-uPAR(－) and pAdeno-X-uPAR(+) respectively. 7 days after transfecting HEK293 cell with linearized pAdeno-X-uPAR(－) and pAdeno-X-uPAR(+), the recombinant adenovirus can be obtained, which were named Ad-uPAR(－) and Ad-uPAR(+) respectively. The virus titre(pfu/ml) of Ad-uPAR(－) was 1.5×10⁸, and the virus titre of Ad-uPAR(+) was 0.5×10⁸. 95D cells were infected with Ad-uPAR(－) and Ad-uPAR(+) in different multiplicity of infection (MOI). Norther blot analysis could detected the expression of antisense and sense RNA for 500 bp fragment of uPAR gene. With the increase of MOI, Western blot analysis indicated that with AD-uPAR(－) infection the protein level of uPAR of 95D cells decreased, and modified Boyden's Chamber assay suggested that the invasive ability of 95D cells also decreased obviously. In 95D cells infected with Ad-uPAR(+), both the mRNA and protein level of uPAR did not decrease, and cells still had high invasive ability. The results indicated that adenovirus is an efficient vector for transferring antisense RNA for uPAR into cells, and antisense RNA for uPAR could obviously inhibit the invasive ability of 95D human lung cancer cells.

Abstract:The amount of GS in Sulfolobus acidocaldarius can be regulated up by different growth medium, this difference is regulated at mRNA level. The GS was purified by DEAE-Sepharose and Sephacryl S-300 to homogeneity. The molecular mass was determined to be dodecameric protein (630 ku) composed of identical subunits of 53 ku. The optical pH of this enzyme is about 7.3, the optical temperature of both γ-glutamyl transferase activity and biosynthetic activity is 90℃, the Arrhenius plots show that the active energy is 47 kJ/(mol·K) for transferase activity and 29 kJ/(mol·K)(40～55℃),10 kJ/(mol·K)(55～90℃) for biosynthetic activity respectively. GS was stable at 78℃ in the presence of Mn²⁺, the K_m values were 3.5 mmol/L, 1.3 mmol/L, 0.5 mmol/L and 0.24 mmol/L for hydroxylamine, glutamine, ADP and Mn²⁺ respectively. The inhibitors experiments showed that the catalytic activity of GS from Sulfolobus acidocaldarius unlike that of others was regulated solely by feed-back inhibition through L-alanine and glycine, the normal inhibitors such as L-tryptophan, L-histidine and 5′-AMP have no inhibitory effect on this enzyme. L-alanine and glycine have shown synergistic effect on catalytic activity of GS. The post-translation modification like other gram positive bcteria is not regulated by adenylylation/deadenylylation system.

Abstract:Human urinary kallikrein (hk-1) and human urinary trypsin inhibitor (hUTI) are two acidic proteins in the urine. Both of them are important drugs. The way of colloid adsorption together with ethyl alcohol precipitation isolated these two proteins successfully. By using of ion-exchange, hydrophobic, affinity chromatography and gel filtration, human urinary kallikrein(hk-1) was purified with single band on SDS-PAGE and single peak on HPLC. The molecular mass was 33 450 u on MALDI-TOF-MS, the pI was about 4.3 on IEF. The influence of pH and temperature on the activity of hk-1 was studied. The whole procedure is suitable for large-scale production.

Abstract:Angiostatin, a 38 ku fragment encompassing the four kringle region of plasminogen, has been identified and characterized to be a potent inhibitor of neovascularization and tumor metastasis. There is a strikingly similarity between tumor metastasis and embryo implantation. However, effect of angiostatin in the mouse blastocyst has never been reported. The results showed for the first time that angiostatin down-regulated the expression of matrix metalloproteinase-2(MMP-2), MMP-9, vascular endothelial growth factor family and its receptor, KDR, and up-regulated the expression of tissue inhibitor of metalloproteinase-1(TIMP-1) and TIMP-2 expression by binding with integrin αVβ3, suggesting that angiostatin may play a role in embryo implantation.

Abstract:Eucommia antifungal protein (EAFP) crystals can be easily grown into big crystals in several hours. By in situ atomic force microscopy (AFM) the dynamic topographic changes were observed on the surfaces of several EAFP crystals and growth rates were measured at different supersaturations of the protein solution. The results of AFM experiments indicated that growth rates of EAFP crystals were strongly and directly related to the supersaturations, in addition to the inherent structural rigidity and the interior stability of the molecule. At higher supersaturation (σ=1.78) the EAFP crystals grew very fast; at moderate supersaturation (σ=1.5) the growth rates were 12 nm/s and 24.2 nm/s along the crystallographic axes b, c of the {100} surface respectively, which were faster than that of lysozyme (6～7 nm/s). Even at lower supersaturation the EAFP crystals grew almost as fast as other protein crystals did. The effects of the concentration of precipitator on crystal growth observed on the crystal growth of AFM at lower supersaturation were also presented.

Abstract:In order to reveal proteins differentially expressed in round-headed sperm in human being, two-dimensional gel electrophoresis and mass spectrometry were performed on 30 normal sperm samples from 10 fertile men and 3 sperm samples from a globozoospermic patient. In the range of molecular weight 7.9～93.5 and pI 4～8, altogether (905±57) and (881±32) spots were detected on the 2-DE map of normal and globozoospermic sperms, respectively. 607 spots were matched between the two groups. Mass spectrometry were performed with 16 proteins which were found to be absent in the round-headed sperms and 1 which expressed in a much lower concentration. Altogether 8 protein spots were identified and their possible roles are discussed. Among them, 3 spots were Golgi apparatus-related, 2 spots were subunits of proteasome and 2 spots were zinc finger proteins. Their absence or down-expression possibly interrupt spermatogenic process.

Abstract:Two genes (treY and treZ) encoding for trehalose-producing enzymes, a maltooligosyl trehalose synthase (MTSase) and a maltooligosyl trehalose trehalohydrolase (MTHase), had been cloned, expressed and sequenced from thermophilic archaebacterium Sulfolobus shibatae B12. The nucleotide sequences of treY and treZ indicated proteins with lengths of 728 and 559 amino acids and molecular masses of 86 ku and 65 ku, respectively. treY and treZ genes from S.shibatae B12 show high sequence homologies with other microbial MTSase and MTHase genes, such as S.solfataricus P2 (93% and 76% identity),S.solfataricus KM1(97% and 95% identity),Sulfolobus acidocaldarius ATCC 33909 (63% and 66% identity),Arthrobacter sp. Q36 (48% and 50% identity),Rhizobium sp. M-11(48% and 52% identity),Brevibacterium sp. (50% and 52% identity). Phylogenetic trees of these enzymes genes were constructed by analysis class of their overall nucleotide sequences. The amino acid sequences of MTSase and MTHase showed identity with members of the glycosyl hydrolase family 13 (α-amylase family). Four important regions highly conserved in the glycosyl hydrolase family 13 exist in the amino acid sequences of all these trehalose-producing enzymes. It is suggested that MTSase and MTHase are members of glycosyl hydrolase family 13 and treY and treZ maybe derived from a common original α-amylase gene.

Abstract:The baculovirus shuttle vector , pBacPAK-IFN-THY was constructed which contains the genes of IFN-α2b and THY-α1. Constructed vector was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The BmN cells were infected with the recombinant virus. The results showed that the protein was successfully prepared and its bioactivities were testified by WISH-VSV system and E-RFC. Results indicated that the expressed fusion protein have bioactivities of both IFN-α2b and THY-α1.

Abstract:In order to explain the reason why brainstem is responsible for the mental retardation in infantile spasms, the maximum energy criterion for burst-event identification in wall turbulence by wavelet analysis was used. It is found that the brainstem-channel of IS patients is very different from that of normal ones in response to stimulation and information transfer. According to the explanation of maximum energy criterion in the turbulence, it was noticed that mental retardation in IS is caused by the block of information transfer in the brainstem. Furthermore, the importance of this method for treatment evaluation is also indicated.

Abstract:Abnormal expression of developmentally important genes leads to inadequate reprogramming development of NT embryo which subsequently causes failure in animal cloning. To identify reprogramming-associated genes in rabbit NT embryo, mRNA differential display has been developed and SPEDDRT-PCR method was set up successfully to overcome the paucity of the biological materials. Using this modified technique, the mRNA content of rabbit NT embryos at different developmental stages (from oocyte to 8～16-cell embryo) were compared, eighty differential displayed bands at different stage in rabbit NT embryo were isolated. A028 amplicon was confirmed by reverse Northern blot. Nucleotide sequences were determined for these cDNAs and database searches identified using NCBI BLAST program. The data suggested that A028 displayed high homology (93%) to CstF3 gene for cleavage stimulation factor, which involved in pre-mRNA 3′-end processing and is required for progression through mitosis. This gene was probably related with preimplantation embryo development, and might play important roles in development of rabbit NT embryo. Of seven organ tissues examined by Northern blot analysis, A028 was only found in ovary. This work paves the way of cloning of the full length of A028 cDNA and further study on gene function.

Abstract:The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. Cytoskeleton system is important for regulating a series of events during meiotic maturation. MⅡ stage mouse oocytes were induced to resume meiosis by parthenogenetic activation, and part of them were treated with cytochalasin B and nocodazole, drugs against cytoskeleton. In order to study functions of microtubules and microfilaments in meiosis, the dynamic changes of microtubules, microfilaments and chromosomes were observed by fluorescent confocal microscopy, and the following results were obtained. Meiotic spindle made up of microtubules is essential to location, separation and movement of chromosomes. The spindle rotation from parallel to vertical with respect to plasmalemma is a premise of polar body extrusion. Microfilaments play a crucial role in regulating spindle rotation. After fulfilling the rotation, microfilaments depolymerize immediately, and therefore do not participate in final extruding of polar body. After forming the pronuclei, microfilaments will assemble again, so as to control the migration of pronuclei.

Abstract:On the basis of T-Rex inducible expression system, a vector that express reporter gene and regulated gene simultaneously were constructed. The induction effects of tetracycline to the gene expression were studied in CV1 cells by transient transfection. An 8.9-fold increase in expression level of reporter gene was observed when CV1 cells, which were transiently transfected with the vector, were induced with tetracycline for 24 h, suggesting that the application of single-plasmid vecor in gene expression induction is also feasible.